Abstract
Male infertility affects 7 % of the male population, and 10 % of infertile men are azoospermic. In these instances, using microsurgical testicular sperm extraction (m-TESE) and intra-cytoplasmic sperm injection (ICSI) helps a significant number of patients. However, in vitro differentiation of diploid germ cells to mature haploid germ cell has the potential to benefit many others, including pediatric cancer survivors who have previously cryopreserved their immature testicular tissue prior to starting gonadotoxic cancer treatment as well as men with spermatogenic arrest. This systematic review evaluates and summarizes half a century of researchers’ efforts towards achieving in vitro spermatogenesis in mammalian species. A myriad of experimental assays and approaches has been developed using whole testis tissue or separated single cells from testis in two- or three-dimensional cell culture systems (2D versus 3D). Recent advances in the mammalian in vitro spermatogenesis, particularly in murine and nonhuman primate systems, hold promise towards translating the availability of in vitro spermatogenesis models in the human clinical setting in the near future.
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Thoma ME, McLain AC, Louis JF, King RB, Trumble AC, Sundaram R, et al. Prevalence of infertility in the United States as estimated by the current duration approach and a traditional constructed approach. Fertil Steril. 2013;99(5):1324–31 e1. doi:10.1016/j.fertnstert.2012.11.037.
Tournaye H. Update on surgical sperm recovery—the European view. Hum Fertil (Camb). 2010;13(4):242–6. doi:10.3109/14647273.2010.522677.
Sadri-Ardekani H, Atala A. Regenerative medicine for the treatment of reproductive system disorders: current and potential options. Adv Drug Deliv Rev. 2015;82-83:145–52. doi:10.1016/j.addr.2014.10.019.
Trowell OA. The culture of mature organs in a synthetic medium. Exp Cell Res. 1959;16(1):118–47.
Steinberger A, Steinberger E, Perloff WH. Mammalian testes in organ culture. Exp Cell Res. 1964;36:19–27.
Steinberger E, Steinberger A, Perloff WH. Initiation of spermatogenesis in vitro. Endocrinology. 1964;74:788–92. doi:10.1210/endo-74-5-788.
Matte R, Sasaki M. Autoradiographic evidence of human male germ-cell differentiation in vitro. Cytologia (Tokyo). 1971;36(2):298–303.
Ghatnekar R, Lima-de-faria A, Rubin S, Menander K. Development of human male meiosis in vitro. Hereditas. 1974;78(2):265–72.
Curtis D. In vitro differentiation of diakinesis figures in human testis. Hum Genet. 1981;59(4):406–11.
Aizawa S, Nishimune Y. In-vitro differentiation of type A spermatogonia in mouse cryptorchid testis. J Reprod Fertil. 1979;56(1):99–104.
Haneji T, Maekawa M, Nishimune Y. In vitro differentiation of Type A spermatogonia from mouse cryptorchid testes in serum-free media. Biol Reprod. 1983;28(5):1217–23.
Toppari J, Brown WR, Parvinen M. Rat spermatogenesis in vitro traced by live cell squashes and monoclonal antibodies. Ann N Y Acad Sci. 1984;438:515–8.
Toppari J, Eerola E, Parvinen M. Flow cytometric DNA analysis of defined stages of rat seminiferous epithelial cycle during in vitro differentiation. J Androl. 1985;6(6):325–33.
Toppari J, Parvinen M. In vitro differentiation of rat seminiferous tubular segments from defined stages of the epithelial cycle morphologic and immunolocalization analysis. J Androl. 1985;6(6):334–43.
Toppari J, Vihko KK, Rasanen KG, Eerola E, Parvinen M. Regulation of stages VI and VIII of the rat seminiferous epithelial cycle in vitro. J Endocrinol. 1986;108(3):417–22.
Haneji T, Koide SS, Nishimune Y, Oota Y. Dibutyryl adenosine cyclic monophosphate regulates differentiation of type A spermatogonia with vitamin A in adult mouse cryptorchid testis in vitro. Endocrinology. 1986;119(6):2490–6. doi:10.1210/endo-119-6-2490.
Tajima Y, Watanabe D, Koshimizu U, Matsuzawa T, Nishimune Y. Insulin-like growth factor-I and transforming growth factor-alpha stimulate differentiation of type A spermatogonia in organ culture of adult mouse cryptorchid testes. Int J Androl. 1995;18(1):8–12.
Hue D, Staub C, Perrard-Sapori MH, Weiss M, Nicolle JC, Vigier M, et al. Meiotic differentiation of germinal cells in three-week cultures of whole cell population from rat seminiferous tubules. Biol Reprod. 1998;59(2):379–87.
Staub C, Hue D, Nicolle JC, Perrard-Sapori MH, Segretain D, Durand P. The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells. Exp Cell Res. 2000;260(1):85–95. doi:10.1006/excr.2000.4998.
Tesarik J, Greco E, Rienzi L, Ubaldi F, Guido M, Cohen-Bacrie P, et al. Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone. Hum Reprod. 1998;13(1O):2772–81.
Tesarik J, Bahceci M, Ozcan C, Greco E, Mendoza C. Restoration of fertility by in-vitro spermatogenesis. Lancet. 1999;353(9152):555–6. doi:10.1016/S0140-6736(98)04784-9.
Suzuki S, Sato K. The fertilising ability of spermatogenic cells derived from cultured mouse immature testicular tissue. Zygote. 2003;11(4):307–16.
Gohbara A, Katagiri K, Sato T, Kubota Y, Kagechika H, Araki Y, et al. In vitro murine spermatogenesis in an organ culture system. Biol Reprod. 2010;83(2):261–7. doi:10.1095/biolreprod.110.083899.
Sato T, Katagiri K, Gohbara A, Inoue K, Ogonuki N, Ogura A, et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature. 2011;471(7339):504–7. doi:10.1038/nature09850. This is the first study that was able to produce functional sperms in vitro from immature mouse testis tissue.
Sato T, Yokonishi T, Komeya M, Katagiri K, Kubota Y, Matoba S, et al. Testis tissue explantation cures spermatogenic failure in c-Kit ligand mutant mice. Proc Natl Acad Sci U S A. 2012;109(42):16934–8. doi:10.1073/pnas.1211845109. This animal study opened a new therapeutic strategy for patients with genetic spermatogenesis defects.
Yokonishi T, Sato T, Komeya M, Katagiri K, Kubota Y, Nakabayashi K et al. Offspring production with sperm grown in vitro from cryopreserved testis tissues. Nat Commun. 2014;5:4320. doi:10.1038/ncomms5320.
Hogg K, Western PS. Differentiation of fetal male germline and gonadal progenitor cells is disrupted in organ cultures containing knockout serum replacement. Stem Cells Dev. 2015. doi:10.1089/scd.2015.0196.
Nagao Y. Viability of meiotic prophase spermatocytes of rats is facilitated in primary culture of dispersed testicular cells on collagen gel by supplementing epinephrine or norepinephrine: evidence that meiotic prophase spermatocytes complete meiotic divisions in vitro. In Vitro Cell Dev Biol. 1989;25(12):1088–98.
Rassoulzadegan M, Paquis-Flucklinger V, Bertino B, Sage J, Jasin M, Miyagawa K, et al. Transmeiotic differentiation of male germ cells in culture. Cell. 1993;75(5):997–1006.
Hofmann MC, Hess RA, Goldberg E, Millan JL. Immortalized germ cells undergo meiosis in vitro. Proc Natl Acad Sci U S A. 1994;91(12):5533–7.
Izadyar F, Den Ouden K, Creemers LB, Posthuma G, Parvinen M, De Rooij DG. Proliferation and differentiation of bovine type A spermatogonia during long-term culture. Biol Reprod. 2003;68(1):272–81.
Sousa M, Cremades N, Alves C, Silva J, Barros A. Developmental potential of human spermatogenic cells co-cultured with Sertoli cells. Hum Reprod. 2002;17(1):161–72.
Tanaka A, Nagayoshi M, Awata S, Mawatari Y, Tanaka I, Kusunoki H. Completion of meiosis in human primary spermatocytes through in vitro coculture with Vero cells. Fertil Steril. 2003;79 Suppl 1:795–801.
Movahedin M, Ajeen A, Ghorbanzadeh N, Tiraihi T, Valojerdi MR, Kazemnejad A. In vitro maturation of fresh and frozen-thawed mouse round spermatids. Andrologia. 2004;36(5):269–76. doi:10.1111/j.1439-0272.2004.00617.x.
Vigier M, Weiss M, Perrard MH, Godet M, Durand P. The effects of FSH and of testosterone on the completion of meiosis and the very early steps of spermiogenesis of the rat: an in vitro study. J Mol Endocrinol. 2004;33(3):729–42. doi:10.1677/jme.1.01493.
Lee DR, Kim KS, Yang YH, Oh HS, Lee SH, Chung TG, et al. Isolation of male germ stem cell-like cells from testicular tissue of non-obstructive azoospermic patients and differentiation into haploid male germ cells in vitro. Hum Reprod. 2006;21(2):471–6. doi:10.1093/humrep/dei319.
Stukenborg JB, Wistuba J, Luetjens CM, Elhija MA, Huleihel M, Lunenfeld E, et al. Coculture of spermatogonia with somatic cells in a novel three-dimensional soft-agar-culture-system. J Androl. 2008;29(3):312–29. doi:10.2164/jandrol.107.002857.
Minaee Zanganeh B, Rastegar T, Habibi Roudkenar M, Ragerdi Kashani I, Amidi F, Abolhasani F, et al. Co-culture of spermatogonial stem cells with sertoli cells in the presence of testosterone and FSH improved differentiation via up-regulation of post meiotic genes. Acta Med Iran. 2013;51(1):1–11.
Xie B, Qin Z, Huang B, Xie T, Yao H, Wei Y, et al. In vitro culture and differentiation of buffalo (Bubalus bubalis) spermatogonia. Reprod Domest Anim. 2010;45(2):275–82. doi:10.1111/j.1439-0531.2008.01281.x.
Hasegawa H, Terada Y, Ugajin T, Yaegashi N, Sato K. A novel culture system for mouse spermatid maturation which produces elongating spermatids capable of inducing calcium oscillation during fertilization and embryonic development. J Assist Reprod Genet. 2010;27(9-10):565–70. doi:10.1007/s10815-010-9442-3.
Lee JH, Oh JH, Lee JH, Kim MR, Min CK. Evaluation of in vitro spermatogenesis using poly(D, L-lactic-co-glycolic acid) (PLGA)-based macroporous biodegradable scaffolds. J Tissue Eng Regen Med. 2011;5(2):130–7. doi:10.1002/term.297.
Abu Elhija M, Lunenfeld E, Schlatt S, Huleihel M. Differentiation of murine male germ cells to spermatozoa in a soft agar culture system. Asian J Androl. 2012;14(2):285–93. doi:10.1038/aja.2011.112. This is the first study that was able to produce sperms in vitro from immature mouse testis-isolated cells. However, fertility potential of sperms was not tested.
Riboldi M, Rubio C, Pellicer A, Gil-Salom M, Simon C. In vitro production of haploid cells after coculture of CD49f + with Sertoli cells from testicular sperm extraction in nonobstructive azoospermic patients. Fertil Steril. 2012;98(3):580–90 e4. doi:10.1016/j.fertnstert.2012.05.039.
Wang P, Suo LJ, Shang H, Li Y, Li GX, Li QW, et al. Differentiation of spermatogonial stem cell-like cells from murine testicular tissue into haploid male germ cells in vitro. Cytotechnology. 2014;66(3):365–72. doi:10.1007/s10616-013-9584-0.
Khajavi N, Akbari M, Abdolsamadi HR, Abolhassani F, Dehpour AR, Koruji M, et al. Role of somatic testicular cells during mouse spermatogenesis in three-dimensional collagen gel culture system. Cell J. 2014;16(1):79–90.
Huleihel M, Nourashrafeddin S, Plant TM. Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta). Asian J Androl. 2015;17(6):972–80. doi:10.4103/1008-682X.154994. This is the first study that was able to initiate spermatogenesis in vitro from juvenile nonhuman primate testis-isolated cells. However, the morphologic study did not show differentiated cell further than round spermatid stage.
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We would like to thank Drs. Stanley Kogan, Bita Nick Kholgh, and Samuel Pendergraft for editorial assistance.
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Guillermo Galdon and Hooman Sadri-Ardekani each declare no potential conflicts of interest.
Anthony Atala is a section editor for Current Urology Reports.
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This article does not contain any studies with human or animal subjects performed by any of the authors.
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Galdon, G., Atala, A. & Sadri-Ardekani, H. In Vitro Spermatogenesis: How Far from Clinical Application?. Curr Urol Rep 17, 49 (2016). https://doi.org/10.1007/s11934-016-0605-3
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DOI: https://doi.org/10.1007/s11934-016-0605-3